FoxP3+ regulatory T-cells (T-reg) are thought to be crucial arbiters of immune tolerance. Unfortunately, protocols that have successfully induced FoxP3+ regulatory T-cells (T-reg) in vitro involve levels of TGFb and IL-2 that would be toxic if administered systemically in vivo. Our preliminary data, however, suggests that it is possible to deliver this same set of T-reg induction cues in a local manner by inculcating TGFb, IL-2 and anti-CD3 Ab into a hydrogel platform made of hyaluronan (HA) and heparin sulfate (HS). This approach roughly doubles the number FoxP3+ cells seen with soluble agents. My hypothesis is that antigen specific Treg can be induced from CD4+FoxP3- precursors using similar HA/HS hydrogels. I propose to investigate this hypothesis in three aims: 1) I will elucidate the mechanisms by which HA and HS in crosslinked form promote Treg induction. 2) I will optimize polyclonal Treg induction using HA/HS hydrogels. 3) I will develop HA/HS hydrogels to induce antigen specific Treg. This work is a culmination of my K08 funded research on HA and the promotion of immune tolerance. This R03 will potentially set the stage for studies on the in vivo and in situ induction of antigen specific tolerance in mouse models of diabetes that I hope to pursue in an R01 application. The significance of the planned work lies in the potential to induce antigen specific tolerance without systemic immune suppression in tissue transplantation and in the treatment of autoimmune diseases. PUBLIC HEALTH RELEVANCE: The treatment of autoimmune disease and organ transplantation frequently involves the administration of systemic immunosuppressants, often leading to infection or unacceptable toxicities. Recent efforts at inducing FoxP3+ regulatory T-cells (T-reg), the body's own mediators of immune tolerance, have faced the same limitations. Our preliminary data, however, suggests that it is potentially possible to locally deliver a set of costimulatory cues previously reported to induce T-reg by using hydrogel constructs made of hyaluronan and heparin sulfate as a platform. In this application I propose to develop hydrogels that are capable of inducing tolerance in an antigen specific manner. The significance of this planned work lies in the potential to induce tolerance to specific autoantigens or transplanted tissues without the need for systemic immunosupression.